In recent years, electrode plates for electrochemical measurements for quantifying a saccharide such as sucrose, glucose contained in blood by a combination of a specific catalytic action of an enzyme and an electronic mediator having an electrode reaction activity have been developed. According to such an electrode plate for electrochemical measurements, after the reaction is allowed between the saccharide and the enzyme, the electronic mediator is electrochemically measured. Then the saccharide contained in the sample solution is quantified indirectly via the electronic mediator.
As one example, the electrode plate for electrochemical measurements is suitable for analyses of solution samples of a slight amount contained in living bodies. Thus, applications of the electrode plate for electrochemical measurements have been attempted to sensors through combining with a variety of organic materials or inorganic materials.
This method is highly specific for the saccharide, accompanied by less influences from the temperature during operation, and the mechanism of the quantifying device is simple; therefore, this method allows ordinary persons to quantify the blood glucose level easily at home.
The electrode response speed of the electrode plate for electrochemical measurements is accelerated as the area of a microelectrode carried by the electrode plate for electrochemical measurements is reduced. Therefore, various electrode shapes, and miniaturization of electrodes have been investigated. However, as the area of the electrode is reduced, the resulting electric current value is lowered. For example, when the area of the electrode is miniaturized to approximately several hundred μm2, detectable electric current value may be lowered to several ten to several nA order. Thus, increase in noise response, and deterioration of the sensitivity may be caused at the time of measurement. Accordingly, in order to avoid these defects, electrode plates for electrochemical measurements in which a plurality of microelectrodes are integrated were studied as in Patent Documents 1 to 4.
Patent Documents 1 to 4 propose methods of producing a large quantity of microelectrodes on a substrate while keeping a constant distance between adjacent microelectrodes with favorable reproducibility.
FIG. 18 shows an overall view (FIG. 18(a)) and a partial enlarged view (FIG. 18(b)) of the construction of a conventional electrode plate for electrochemical measurements disclosed in Patent Document 1. The electrode plate for electrochemical measurements 200 is constructed by laminating an insulative substrate 201, a bottom electrode body 202 that functions as an oxidation electrode, an insulating layer 203, and a surface electrode 204 that functions as a reduction electrode. A large number of cylindrical micropores 205 are formed on the surface of the surface electrode 204, and the film face of the bottom electrode body 202 is exposed to the micropore 205.
The insulative substrate 201 is composed of, for example, a silicon substrate with an oxide film, generally referred to, in which oxide film 201b is adhered on the main surface of silicon substrate 201a. The bottom electrode body 202 is an oxidation electrode formed with a metal, a semimetal, a carbonic material, or a semiconductor on the surface of the oxide film 201b of the substrate 201 (i.e., insulator surface). The surface electrode 204 is a reduction electrode formed with a metal, a semimetal, or a semiconductor on the insulating layer 203, similarly to the bottom electrode body 202.
A working electrode pair, i.e., paired oxidation electrode and reduction electrode, is formed with an exposed part of the bottom electrode body 202 from micropore 205 (hereinafter, referred to as oxidation electrode 202a), and the surface electrode 204. In other words, both the oxidation electrode 202a and the surface electrode 204 function as working electrodes, and more specifically, the exposed part of the bottom electrode body 202 functions as an oxidation electrode, while the surface electrode 204 functions as a reduction electrode, as described above. In FIG. 18, the reference numeric character 206 represents an opening for drawing the electrode, opened so as to connect an outer lead to one end of the bottom electrode body 202. Herein, the micropore 205 represents a hole that completely penetrates through the insulating layer 203 and the surface electrode 204, and then reaches to the surface of the bottom electrode body 202.
In an apparatus for electrochemical measurements in which the electrode plate for electrochemical measurements as described above is used, a potential is applied to the bottom electrode body 202 and the surface electrode 204 for achieving an electric current response. When the apparatus for electrochemical measurements is constructed with three electrodes, i.e., the oxidation electrode body 202a, surface electrode 204, and a counter electrode (not shown in the Figure), a potential is applied between the oxidation electrode body 202a and the counter electrode, and between the surface electrode 204 and the counter electrode, provided that the potential shown by the counter electrode in the sample solution is zero.
In addition, when the apparatus for electrochemical measurements is constructed with four electrodes, i.e., the oxidation electrode body 202a, surface electrode 204, a reference electrode (not shown in the Figure), and an auxiliary electrode (not shown in the Figure), a potential is applied between the oxidation electrode body 202a and the reference electrode, and between the surface electrode 204 and the reference electrode, provided that the potential shown by the reference electrode in the sample solution is zero.
Patent Document 4 and Nonpatent Document 1 propose an electrode plate for electrochemical measurements in which cylindrical micropores 205 are provided such that the intervals among them become greater than their diameter, and report the results of electrochemical measurements using the same. In these Documents, the surface electrode 204 that is a macroelectrode has an area greater than the bottom electrode that is an assembly of microelectrodes. At the time of measurement, potentials are applied, respectively, which can cause an oxidative reaction on the oxidation electrode body 202a, and a reductive reaction on the surface electrode 204. It is reported that self-induced redox cycle is thus generated between the oxidation electrode body 202a and the surface electrode 204, and then apparently high electric current response can be achieved.
In this manner, a target substance such as a saccharide is quantified via an electronic mediator that is present in a sample solution. Alternatively, even though a potential that causes a reductive reaction is applied on the oxidation electrode body 202a, while a potential that causes an oxidative reaction is applied on the surface electrode 204, similar self-induced redox cycle is generated.
Hereinbelow, the self-induced redox cycle described in Patent Document 4, and Nonpatent Documents 1 and 2 is explained with reference to FIG. 19.
The self-induced redox cycle in FIG. 19 proceeds on two working electrodes, i.e., microelectrode 221 and macroelectrode 222. An oxidative reaction of reductant 224 is caused to produce oxidant 225 on the surface of the microelectrode 221, and then the oxidation current flows to the microelectrode 221. On the surface of a part 222a, which is close to the microelectrode 221, of the macroelectrode 222, the oxidant 225 is reduced to be converted into reductant 226, and then the reductive electric current flows to the macroelectrode 222. Furthermore, the reductant 225 is diffused to reach to the surface of the microelectrode 221, and then an oxidative reaction is caused again from the reductant 224 to the oxidant 225, and oxidation current flows toward the microelectrode 221.
As a consequence, the reductant 224 can be fed to the surface of the microelectrode 221 by reducing the oxidant 225 generated from the microelectrode 221 to give the reductant 226 on the surface of the macroelectrode 222a. Accordingly, as a result of occurrence a so-called redox cycle reaction in which an oxidative reaction and a reductive reaction recur between the microelectrode 221 and the macroelectrode 222a, an electric current constantly flows to the microelectrode 221, and thus the target substance contained in a sample solution in a slight amount can be detected and quantified. Moreover, in order to improve the efficacy of the measurement with high sensitivity, electrode pairs consisting of an oxidation electrode and a reduction electrode by which a redox cycle proceeds are formed as many as possible through forming a larger number of the microelectrodes 221 on the substrate.
Patent Document 1: Japanese Patent Publication No. 2556993 (page 6, FIG. 1)
Patent Document 2: Japanese Patent Publication No. 2564030 (page 7, FIG. 2)
Patent Document 3: Japanese Patent Laid-Open Publication No. 2006-78404 (page 25, FIG. 1)
Patent Document 4: Japanese Patent Publication No. 3289059 (page 16, FIG. 5)
Patent Document 5: Japanese Patent Laid-Open Publication No. 2007-010429 (FIG. 3, FIG. 4)
Nonpatent Document 1: J. Electrochem. Soc., Vol. 138, No. 12, page 3551
Nonpatent Document 2: Koichi Aoki et al., “Electrochemical Measurement Method Using Microelectrode” edited by The Institute of Electronics, Information and Communication Engineers, published on Feb. 10, 1998 pages 48-49 and 70-71